小麦全蚀病菌病毒的研究

 自山东泰安及烟台地区的小麦全蚀病菌中提取到两种类型颗粒,一类是球形至多边形的病毒颗粒,直径在22~45毫微米之间,多数为27~32毫微米;另一类是棒状颗粒,长度为40~185毫微米×16毫微米,平均108×16毫微米,提取的球形病毒具有核蛋白的典型吸收光谱,最大和最小吸收值的波长分别在260毫微米和240毫微米,A260/240=1.24,A260/280=1.62。以烟台株病毒制备的抗血清,分别与烟台及泰安株病毒在琼脂免疫双扩散中作用均产生沉淀线,表明二者具有共同的血清型。烟台株病毒经苯酚抽提后的核酸样品最大吸收波长是260毫微米,最小是230毫微米,经聚丙烯酰胺-琼脂糖凝胶电泳分离为两条迁移率相近的带,表明此病毒有两个基因片断,在分类上属于需要两个dsRNA片断才能复制的等轴dsRNA真菌病毒。其外壳蛋白经测定含分子量70,000的多肽。<>br从浙江、湖北等11个省(市)的12株全蚀病菌中均提取到了病毒,比较了各菌的菌落颜色,生长速度和病毒浓度。     

Analysis of natural populations and possible natural reassortment of Cucumber mosaic virus

Plants naturally infected with Cucumber mosaic virus (CMV) were collected and analyzed by electrophoresis of the replicative form of dsRNA and by Northern blot hybridization using CMV RNA-specific probes. Some of the CMV-infected plants, especially winter crops, contained two kinds of RNA 1 segments or RNA 2 segments (or both), suggesting that mixed infections of CMV occurred naturally. Single-aphid-transmitted isolates (SATIs) from the field isolate containing two RNA 1 segments were grouped into three types by the electrophoretic mobility of RNA 1 (i.e., those containing one slow segment, those containing one fast segment, and those containing both). Furthermore, SATIs and single-lesion isolates, generated from the plants inoculated with a mixture of two CMV isolates that could be differentiated by their electrophoretic dsRNA profiles, were analyzed by dsRNA, indicating that nonparental progenies were observed. These results suggested that genetic reassortment of CMV RNA may occur in nature and that this is an important mechanism in CMV evolution.     

Nucleotide sequence and symptom modulating analysis of a Peanut stunt virus-associated satellite RNA from Poland: high level of sequence identity with the American PSV satellites

Two peanut stunt virus isolates from Poland (PSV-Ag and PSV-P) have been studied. The isolates produce similar systemic symptoms onNicotiana tabacum plants but the symptoms onN. benthamiana, Pisum sativum andDatura stramonium plants are much stronger for the PSV-P isolate. Analysis of the RNA extracted from purified virions by gel electrophoresis and RT-PCR amplification allowed the detection of a satellite RNA in the PSV-P isolate. The nucleotide sequence of this European PSV satellite was determined and found to have a high degree of identity with the sequences of the four American PSV satellites previously studied, which were found to have either no effect or ameliorate the PSV symptoms in tobacco plants (Collmer et al., 1985; Naidu et al., 1991). The possible role of the European PSV satellite in the modulation of viral symptoms has been studied but no effect was observed when the purified satellite was used with the PSV-Ag isolate as helper virus on any of the three hosts cited above.     

A third cryptic virus in beet (Beta vulgaris)

Japanese leaf beet Beta vulgaris var. cicla cv. Fudanso plants were found to contain four double-stranded RNA (dsRNA) components in apparently healthy beet plants. Two were identified as from beet cryptic virus 1 (BCV1), but the other two showed different mobilities on gel electrophoresis and were transcribed into complementary DNA (cDNA) and cloned. Hybridization analysis showed no significant sequence homology between these two dsRNAs and the dsRNA components of BCV1 or the other known cryptic virus of beet, BCV2. Slot- and dot-blot hybridization were used with cDNA clones as probes to identify plants containing these two dsRNA components. Virus particles were purified from these plants and were shown to contain the two new dsRNA components, thus demonstrating the existence of a new beet cryptic virus, which we have called BCV3.     

北京地区侵染茄子的番茄斑萎病毒分子鉴定与分析

调查发现北京地区一温室栽培茄子Solanum melongena L.出现严重病毒病。利用基于小RNA的高通量测序技术和RT-PCR方法,明确了引起茄子病害的病毒种类为番茄斑萎病毒,将其命名为TSWV-eggplant分离物。进一步克隆了该病毒的基因组全长(S RNA、M RNA、L RNA),并构建其系统发育树。结果表明,该分离物的S RNA与美国分离物亲缘关系较近,M RNA与中国分离物亲缘关系较近,而L RNA与韩国分离物亲缘关系较近。因此,本研究发现的TSWV分离物与国内已发生报道的分离物不同,该分离物是否存在不同分离物之间基因组的重组需要进一步研究。     

RNA/cDNA hybridization studies of UK isolates of barley yellow mosaic virus

The RNA components of two isolates of BaYMV from Streatley and Wiltshire were analysed by agarose gel electrophoresis. Northern blots of extracts of both isolates analysed with random-primed cDNA probes to RNA1 and 2 of the Streatley isolate showed little or no sequence homologies between the two isolates, confirming other evidence that they should be considered as different viruses.     

中国水仙上一种球形病毒的研究Ⅱ.病毒提纯及其生化特性

 繁殖于昆诺阿藜(Chenopodium quinoa)、千日红(Gompherena globosa)及番杏(Tetragonia.expansa)上的病毒分离物,经PEG6000沉淀结合差速离心浓缩后,用Sepharose 4B柱层析法进行分离纯化,获得保持侵染活性的病毒纯化物;而且表明,用昆诺阿藜繁殖病毒,效果最优。紫外扫描测定,纯化病毒的最高吸收波长为258nm,最低吸收波长为244nm,A260/A280为1.515。分析超速离心测定,病毒粒体为单一沉降组分,S20.w约73S。SDS-PAGE结果表明,病毒衣壳蛋白亚基由一条分子量为27.88×10³±2.99×10³daltons的多肽组成。根据紫外吸收估计,病毒粒体中核酸含量约12-15%;RNaseA酶解及变性处理病毒核酸后琼脂糖变性电泳结果说明,病毒基因组为一条ssRNA。根据该病毒的生物学特性及生化特性,初步确认为一种新的病毒分离物,并定名为中国水仙条纹病毒(Chinese Narcissus Strip Virus,ChNSV),其密码程式为R/1:*/12-15:S/S:S/*。     

Differences in cultivar response and complete sequence analysis of two isolates of wheat yellow mosaic bymovirus in China

Seeds of selected European and Japanese winter wheat cultivars were grown at two experimental sites in China, namely Yaan, Sichuan province (YA), and Yangzhou, Jiangsu province (YZ), where wheat yellow mosaic bymovirus (WYMV) was severe. There were some differential responses of the cultivars to the virus isolates present at the two sites. The complete nucleotide sequence of both RNAs of both virus isolates was determined. Their genome organization was identical to that reported for a Japanese isolate and the sizes were very similar. Nucleotide comparisons demonstrated that parts of the CI and NIa coding regions on RNA1 and the N-terminal part of the P2 coding region on RNA2 were particularly variable, while substantially conserved regions occurred in the 3' UTR of RNA2, the 7K, one part of the CI and parts of the NIb and coat protein. It seems unlikely that differences in the 7K and NIa-VPg proteins are responsible for virulence differences and the CI and NIb regions were considered the most promising for further study.     

瓜类褪绿黄化病毒山东分离物全基因组序列扩增及分析

为明确分离自山东省寿光市甜瓜上的瓜类褪绿黄化病毒(cucurbit chlorotic yellows virus,CCYV)分离物的全基因组序列信息和遗传变异情况,利用毛形病毒属Crinivirus简并引物进行RTPCR检测,利用RACE技术结合RT-PCR方法克隆CCYV山东分离物2条RNA链的全基因组序列,通过与GenBank中其它地区CCYV分离物的全长序列进行比对分析其同源性,并基于CP基因序列构建系统进化树分析其遗传变异情况。结果表明,山东分离物经RT-PCR检测和测序后确定为CCYV。CCYV山东分离物与其它CCYV分离物的RNA1链和RNA2链的全基因组序列一致性的平均值分别为99.82%和99.88%,且2条链的5′末端均比较保守,没有碱基突变的情况发生;RNA1链3′末端存在2个碱基变异,RNA2链3′末端存在1个碱基变异。CCYV不同地区分离物主要分为3个簇群,其中山东分离物和中国其它地区分离物、日本分离物、苏丹分离物、黎巴嫩分离物和塞浦路斯分离物聚类在一起。研究表明CCYV基因组序列比较保守,该病毒的分化可能与地理来源存在一定的相关性。     

Molecular Variability of the Capsid Protein of the Prune Dwarf Virus

Sequences of the capsid protein gene and the preceding intergenic region of eleven isolates of prune dwarf virus from central Europe were determined. The isolates were obtained from plum, cherry and peach trees. Comparison of all sequenced isolates (including two sequences published previously) revealed high (88%) conservation of the capsid protein gene. The highest degree of identity was observed in the C-terminal half, where only 13 amino acid substitutions could be observed in contrast to the N-terminal half with 22 substitutions. No reasonable correlation between amino acid substitutions and host species and/or geographic origin of the isolates was observed. Alignment with capsid protein genes of other ilarviruses revealed apple mosaic virus, elm mottle virus, lilac ring mottle virus and prunus necrotic ringspot virus as the most related to prune dwarf virus. Unlike the isolates of related prunus necrotic ringspot virus all the isolates of prune dwarf virus shared extensive conservation of the intergenic region. Portions of RNA3 were selected for design of universal primers for PCR detection.     

Attenuation of Virulence in Sclerotinia homoeocarpa during Storage is Associated with Latent Infection by Ophiostoma mitovirus 3a

The hypovirulence-associated mitovirus, Ophiostoma mitovirus 3a (OMV3a), has been shown to be widespread in eastern Canadian populations of Sclerotinia homoeocarpa in the form of latent infection. Latent infection by OMV3a was not associated with an apparent phenotype and did not significantly reduce the growth and virulence of the pathogen. In the present study, we found that isolates of S. homoeocarpa latently infected by OMV3a can change to hypovirulent isolates after storage at 4 °C, and that this attenuation of virulence was associated with increased concentration of the OMV3a virus. Recurrent observations revealed that up to 29.8% of latently infected isolates changed to hypovirulent isolates after 21 months of storage. Transmission of OMV3a dsRNA from latently infected isolates to virus-free isolates resulted in latent infection of the recipient isolates, indicating that latent infection by OMV3a was not associated with genetic differences in the fungal host. The RNA genomes of the OMV3a virus in an isogenic pair of latently infected and hypovirulent isolates were sequenced and compared. Each of the two RNAs contained an open reading frame of 726 amino acids with conserved motifs typical of RNA-dependent RNA polymerase (RdRp). The OMV3a RNA sequences in these two isolates share 95.1% nucleotide and 94.6% amino acid sequence identities. The development of hypovirulence from latent infection by OMV3a virus may provide new strategies to improve the biological control efficacy of hypovirulence in dollar spot management.     

Effect of Diaporthe RNA virus 1 (DRV1) on growth and pathogenicity of different <Emphasis Type="Italic">Diaporthe</Emphasis> species

A 4.1 kbp positive-strand RNA virus known as Diaporthe RNA virus 1 (DRV1) occurs in hypovirulent, non-sporulating isolates of the fungal pathogen Diaporthe perjuncta. A full-length cDNA clone of DRV1 was developed and RNA transcribed from the cDNA clone used to transfect different Diaporthe spp. The transfected species included three D. ambigua isolates and an unidentified Phomopsis asexual state of a Diaporthe sp. Successful transfections were confirmed using RT-PCR. Although the in vitro-transcribed positive sense single-stranded RNA used for transfection included vector sequences at both ends, the genomes of progeny virus from DRV1-transfected isolates were free of the vector sequences. Transfection resulted in morphological changes in these fungal pathogens. However, the presence of DRV1 did not reduce growth rate in two of the three D. ambigua or the Phomopsis sp. significantly. Pathogenicity studies showed that the transfected isolates have reduced aggresiveness.     

Molecular and biological characterization of Potato mop‐top virus (PMTV,Pomovirus) isolates from the potato‐growing regions of Colombia

Potato mop‐top virus (PMTV) causes necrotic flecks inside and on tubers in temperate countries. In South America, these symptoms have not been observed, although the presence of the virus has been confirmed in the Andes and in Central America. To characterize PMTV isolates from the Andes, soil samples were taken from the main potato‐producing regions in Colombia and virus was recovered by planting Nicotiana benthamiana as bait plants. The complete genomes of five isolates were sequenced and three of the isolates were inoculated to four different indicator plants. Based on sequence comparisons, three types of RNA‐CP (RNA2) and RNA‐TGB (RNA3) were found. The isolates from the centre of the country (CO3 and CO4) were similar to isolates from Europe. The genomes of CO1, CO2 and CO5 differ from other PMTV isolates, placing them in a separate clade in phylogenetic trees. The three Colombian isolates (CO1, CO2 and CO5) only induced slightly different symptoms in the indicator plants. However, the isolate from the northwest of the country (CO1) induced stronger symptoms in N. benthamiana including severe stunting. A correlation between the genotype of the isolates and the symptoms they induced on indicator plants was not found.     

Molecular and serological diversity in <Emphasis Type="Italic">Apple chlorotic leaf spot virus</Emphasis> from sand pear (<Emphasis Type="Italic">Pyrus pyrifolia</Emphasis>) in China

Apple chlorotic leaf spot virus (ACLSV) isolates from sand pear (Pyrus pyrifolia) were characterized by analyzing the sequences of their coat protein (CP) genes and serological reactivity of recombinant coat proteins (rCPs). The sequences of CP genes from 22 sand pear isolates showed a high divergence, with 87.3–100% identities at the nucleotide (nt) level and 92.7–100% identities at the amino acid (aa) level. Phylogenetic analysis on the aa sequence of CP showed that the analyzed ACLSV isolates fell into different clusters and all isolates from sand pear were grouped into a large cluster (I) which was then divided into two sub-clusters (A and B). Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot and enzyme-linked immunosorbent assay (ELISA) analyses demonstrated that rCPs of eight ACLSV isolates (PP13, PP15-2, PP24, PP43, PE, PP54, PP56 and ACLSV-C) from two sub-clusters had different mobility rates and serological reactivity. The rCPs of five isolates grouped into the sub-cluster A showed stronger reactivity with antibodies against rCPs of a sand pear isolate ACLSV-BD and virions of a Japanese apple isolate P-205 than that with the antibody against a Chinese apple isolate ACLSV-C. Three isolates grouped into the sub-cluster B showed stronger reactivity with the antibody against ACLSV-C. The antigenic determinants of CPs from these eight isolates and isolates ACLSV-BD and P-205 were predicted. These results contribute to a further understanding of molecular diversity of the virus and its implication in serological detection.     

Molecular variability of Spanish and Hungarian isolates of Tomato torrado virus

The population structure and genetic variation of Tomato torrado virus (ToTV) were estimated from 19 Spanish isolates collected from 2001 to 2009 in different tomato‐production areas by analyses of the partial nucleotide sequences of five regions of the virus genome: the protease cofactor (Pro‐Co) and the RNA‐dependent RNA polymerase (RdRp) in RNA1, and the movement protein (MP) and two subunits of the coat protein (CP; viz. Vp35 and Vp23) in RNA2. Three Hungarian isolates of the virus were also included in the analyses. All the ToTV isolates clustered together in the phylogenetic analysis of the nucleotide sequences of the different regions. However, some genetic diversity was observed in the case of the two CP subunits among the Gran Canaria isolates and the remaining ToTV‐isolates analysed, which grouped together. A high similarity was observed among all the isolates and the two published ToTV isolates: the ToTV type isolate (PRI‐ToTV0301) and the Polish isolate Wal03. The most variable encoding regions studied were those on RNA2. In general, no correlation was found between genetic diversity and collection date. Studying the genetic distances between pairs of sequences, the ratio between nonsynonymous (amino‐acid‐replacing) and synonymous (silent mutational) substitutions was low, indicating a strong negative selective pressure in the studied regions. Nine negatively selected sites (distributed in Pro‐Co, MP, Vp23 and Vp35) and just one positively selected one (in Pro‐Co) were found for all the genome regions studied.